The broad goal of this renewal proposal is to define the basic principles of CFTR channel gating in the context of a new allosteric gating model. CFTR channel dysregulation causes multiple human disorders including cystic fibrosis and secretory diarrhea. Like other ABC transporters, CFTR has two nucleotide binding domains (NBDs) that bind ATP in pockets at the interface of an NBD dimer. CFTR also has a unique regulatory domain (R domain) that inhibits channel opening unless phosphorylated by protein kinase A (PKA). How ATP binding to the NBDs and phosphorylation of the R domain change the conformation of the pore-forming transmembrane domains (TMDs) to mediate channel opening is unknown. During the current funding period we made several significant discoveries that shed new light on the CFTR gating mechanism;notably;(i) cytosolic loops 1 and 3 appear to form a critical interface along the CFTR symmetry axis that conformationally links ATP binding at the NBDs to pore opening at the TMDs;(ii) certain point mutations in these loops promote constitutive (ATP-independent) channel opening and rescue the defective gating of a common CF mutant channel (G551D);(iii) the existing CFTR gating data are well described by an allosteric model in which ATP and ADP function as agonist and inverse agonist, respectively and (iv) the R domain regulates channel isomerization (i.e., agonist-independent channel opening) independently of ATP binding or NBD dimerization, possibly by directly interacting with the cytosolic loops. These findings set the stage for the next phase of our project in which we will pursue three specific aims. Aim 1: Determine how cytosolic loops 1 and 3 regulate CFTR channel isomerization downstream of ATP binding. Aim 2: Test and refine an allosteric gating model in which ATP and ADP function as agonist and inverse agonist, respectively. Aim 3. Define the precise mechanism by which phosphorylation of the R domain regulates CFTR channel opening. This project should significantly improve our understanding of the basic principles of CFTR channel gating, and may lead to new approaches for treating CFTR-related diseases.